1. Field of the Invention
This invention provides methods and compositions related to the oral administration of zymogen forms of enzymes for therapeutic purposes. The invention therefore relates to the fields of biology, molecular biology, chemistry, pharmacology, and medicine.
2. Description of Related Disclosures
The oral delivery of proteins or enzymes to the gastrointestinal (GI) tract is problematic as a result of poor protein stability due to both gastric pH and proteolytic digestion by GI enzymes as well as poor absorption. A natural solution to aid protein stability is the use of proenzymes or zymogens (these terms are used interchangeably herein to refer to an inactive precursor of an enzyme) rather than the active enzyme. Zymogens may be more stable than the corresponding enzyme itself, as when, for example, an enzyme can cleave itself into inactive fragments. The activation process of a zymogen may involve, for example, a change to the active site or exposure of an active site through cleavage of a peptide bond. There are abundant examples of zymogens in nature, including pepsinogen, trypsinogen, chymotrypsinogen, prolipase, propapain, many caspases, some lipases and certain amylases, and many members of the coagulation and complement system.
Other zymogens of interest include certain cysteine proteases. For example, cysteine endoprotease (EP) B2 from barley is a zymogen. This barley derived protease and other similar proteases derived from the germinating seeds of the gluten-containing cereals have been identified as effective agents for the detoxification of gluten, the causative agent in Celiac sprue and dermatitis herpetiformis (see U.S. Pat. No. 7,303,871, incorporated herein by reference). A modified, recombinant form of the barley-derived zymogen called “ALV001” (the active form of this enzyme is termed “ALV001* herein) has been used as part of a combination enzyme therapy (including a prolyl endopeptidase (PEP), such as Sphingomonas capsulata PEP) for oral administration to Celiac sprue and dermatitis herpetiformis patients to aid in the digestion of gluten before it can exert its toxic effects in these patients (see U.S. Pat. No. 7,320,788; U.S. Patent Application Publication No. 20080193436; PCT Patent Publication No. 2008/115428; PCT Patent Publication No. 2008/115411; and PCT patent application US2009/004791, each of which is incorporated herein by reference). The ALV001 zymogen is inactive and becomes active (converts to ALV001*) below pH 5 but is not activated at a higher pH. Pepsinogen, another zymogen of a cysteine protease, depends on gastric acidity to be activated to pepsin. The reference Janabi et al. (J. Biol. Chem, 247, 4626-4632, 1972) reports that the first-order catalysis of pepsin generation below pH 3 is rapid and intramolecular while it is lower and predominantly intermolecular above pH 4. Propapain, a third example of a proenzyme of a cysteine protease, is activated by an intramolecular rearrangement of the thiol disulfide active site.
Achieving therapeutic efficacy via oral administration of a zymogen can be a balancing act between the stability of the protein, with its rapid degradation by gastric acidity and proteolysis by GI enzymes, and activation to the active enzyme form. Prior to administration, stability of the protein during storage can be aided by use of a more stable zymogen form. Gastric pH in the fasting state is typically quite acidic in the range of pH 2 (between 1.5 and 2.8) and can be raised in the postprandial state to a pH in the range of 4 to 5 or even higher, and then may return to acidic pH within 45 minutes to 1 or 2 hours, depending on the buffering capacity of the meal. With the window and variation of postprandial pH, a zymogen may not be activated to the enzyme form optimally or at all, as they are subjected concurrently to rapid degradation in the stomach. A dosage form that can provide optimal timing for the activation of the zymogen to maximize its activity and duration of activity in the stomach can dramatically improve the efficacy of the therapy intended by administration of that dosage form.
A number of dosage forms that vary release or stability with pH have been discussed in the art. U.S. Pat. No. 4,601,896 describes a capsule consisting of collagen and an activated collagenase to protect therapeutic agents and specifically digestive enzymes from degradation by gastric acidity. The enzymes are reportedly spared degradation in the stomach by delaying their release from the dosage form until it reaches the intestine. Enteric coatings and alkaline buffers were cited as other approaches to avoid gastric degradation. U.S. Pat. No. 5,316,772 and U.S. Patent Application Publication No. 20070154547 describe the use of pH dependent enteric coatings to deliver a drug to different regions of the GI tract.
A number of other dosage forms from which drug release is triggered by changes in pH have been described. U.S. Pat. No. 5,609,590 describes an osmotic bursting device in which the release of drug is triggered by a pH sensitive coating in the range of pH 3 to 9. U.S. Pat. No. 6,068,853 describes a transdermal dosage form where the driving force for drug release oscillates with pH, and the system is manually activated by mixing the contents of two reservoir compartments prior to application to the skin. U.S. Patent Application Publication No. 20050244504 describes pH triggered particles for delivering agents into an acidic environment, in particular, intracellularly in lysosomes. U.S. Pat. No. 7,399,772 describes the use of buffer with proton pump inhibitors for providing stability in the GI tract. U.S. Patent Application Publication No. 20070196491 describes the addition of an organic acid to an immediate release dosage form to improve the solubility of poorly soluble weak basic drugs with an aqueous solubility at pH 6.8 of less than 200 μg/ml. The patent reports that the addition of a free acid to a poorly soluble basic drug may be used to modify its rate of release.
There remains a need for pharmaceutical formulations and unit dosage forms that provide not only improved stability of a zymogen in the stored dosage form but also rapid activation of the zymogen in the fed stomach. The present invention meets this and other needs.